Coxiella burnetii is an obligate intracellular bacterial pathogen that is typically acquired through aerosol exposure. It is the etiologic agent of acute Q fever and chronic diseases such as endocarditis, hepatitis, and chronic fatigue. Acquired by inhalation, C. burnetii initially contacts alveolar macrophages and undergoes a bi-phasic life cycle involving structurally distinct forms of the pathogen. Infection is thought to normally be initiated by the metabolically inactive, environmentally stable small cell variant (SCV) followed by conversion to the replicative, metabolically active, large cell variant (LCV). After infecting the host cell, C. burnetii replicates in vacuoles that retain many of the features of mature phagolysosomes. The molecular mechanisms used by C. burnetii to parasitize their host cells are largely unknown. The genome of C. burnetii (Nine Mile Phase I strain) revealed genes homologous to the type IV secretion system (TFSS) of Legionella pneumophila, suggesting that C. burnetii possesses a specialized secretory pathway for interacting with host cells. However, little is known about the TFSS of C. burnetii or its possible role during the infectious cycle. Our central hypothesis is that the C. burnetii T4SS expression and structure/function changes during SCV to LCV conversion. The goals of the ongoing research proposed in this application are to (i) characterize and define the expression of C. burnetii T4SS genes during the conversion of the bacteria from SCVs to LCVs and, (ii) determine the structure and protein interactions within the C. burnetii T4SS with particular emphasis in differences between the SCV and LCV forms of the pathogen. We will test our central hypothesis by accomplishing the following Specific Aims: Aim 1. Define the expression of T4SS genes during the conversion of C. burnetii from SCVs to LCVs after infection of host cells. We will use RT-qPCR to define the temporal expression of C. burnetii T4SS genes during the first 36 hpi following a low speed centrifugation initiated infection and Indirect Fluorescent Antibody (IFA) and Immuno-electron (IEM) microscopy to define and differentiate the C. burnetii T4SS expression on small cell variant (SCV) and large cell variant (LCV) forms of the pathogen during this time period. Aim 2. Determine the sub-cellular localization, structure, and protein-protein interactions of the C. burnetii T4SS in SCVs and LCVs. We will use antibodies against C. burnetii T4SS proteins in immunoblot analysis of bacterial fractions, as well as IFA and IEM analysis of C. burnetii grown in cell-free media to define T4SS sub-cellular localization, protein-protein interactions, and SCV/LCV specificity. Understanding the virulence mechanisms employed by this unique pathogen to survive within the harsh environment of the host cell phagosome and cause disease will enable us to develop countermeasures to this poorly understood bacteria.